High levels of serum cholesterol are a significant causative effect in the pathogenesis of atherosclerosis and associated diseases such as atherosclerotic coronary heart disease, atherosclerotic cerebral vascular disease, renal disease, etc. It is also believed that lowering of blood cholesterol levels is associated with amelioration of atherosclerotic vascular diseases (Goodman, D. S. et al., Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Arch. Intern. Med. 148:36-69, 1988; Kromhout, D. et al., Serum cholesterol and 25-year incidence of and mortality from myocardial infraction and cancer. (See The Zutphen Study. Arch. Intern. Med. 148:1051-1055, 1988.) In 1984, a National Institutes of Health consensus development conference panel recommended a framework of detection and treatment of hypercholesterolemia. Based on this study, the National Cholesterol Education Program has made the well-known recommendation to adults: "Know your cholesterol number" (Luepker, R. V. et al., Recommendations regarding public screening for measuring blood cholesterol. Summary of a National Heart, Lung, and Blood Institute Workshop, October 1988. Arch. Intern. Med. 149:2650-2654, 1989).
The conventional methods recommended for achieving reduced serum cholesterol levels are through reduction of dietary intake of cholesterol and other fats, and treatment of hypercholesterolemic individuals with drugs designed to lower blood cholesterol. The blood cholesterol levels are particularly associated with homeostatic mechanisms related to levels of plasma lipoproteins that serve as carriers of cholesterol. The so-called dangerous lipoproteins, from the standpoint of atherosclerotic risk, are the low density lipoproteins ("LDL"). The levels of LDL are regulated by the functional activities of LDL receptors on the surfaces of cells, particularly in the liver (see Brown, M. S. and Goldstein, J. L. A receptor-mediated pathway for cholesterol homeostasis. Science 232:34-47, 1986). Many of the strategies for using drugs to reduce blood cholesterol involve interference with the processing of cholesterol derived from LDL (Brown and Goldstein, 1986). The extent that cholesterol can be reduced by diet is limited by numerous factors, and the reduction of cholesterol by drugs is often associated with unwanted side effects. In any case, a variety of additional variables, such as genetic background, stress, and age, can influence cholesterol levels. Additional methods for reduction of cholesterol would be expected to have beneficial health effects, particularly in individuals who receive such treatment before significant progression of atherosclerotic disease has occurred.
To our knowledge, humans have never been actively immunized against cholesterol. The safety of active immunization against cholesterol, as well as the potential consequences relating to serum cholesterol levels or progression of atherosclerosis due to intake of dietary lipids, has not been established. It has been demonstrated that human sera usually do contain varying quantities of "naturally-occurring" antibodies to cholesterol, depending on the individual serum (See Alving et al., Naturally occurring autoantibodies to cholesterol in humans. Biochem. Soc. Trans. 17:637-639 (1989)). However, there has not been any correlation of such naturally-occurring antibodies with serum cholesterol levels or with atherosclerosis.
The possibility has been discussed that naturally-occurring antibodies to cholesterol might be a normal part of the aging process and might contribute to (rather than ameliorate) atherosclerosis (Alving, C. R. Antibodies to liposomes, phospholipids, and cholesterol: Implications for autoimmunity, atherosclerosis, and aging. In: Horizons in Membrane Biotechnology, Nicolau, C. and Chapman, D., editors, Wiley-Liss, pp. 40-41, 1990).
Although the inventors have not found any prior art teaching the immunization of humans with cholesterol, in the literature there has been one description of an attempt to ameliorate hypercholesterolemia and atherosclerosis in rabbits by immunological means. Bailey et al. immunized rabbits with an antigen consisting of cholesterol conjugated to bovine serum albumin (See Bailey et al., Immunization with a synthetic cholesterol-ester antigen and induced atherosclerosis in rabbits. Nature 201:407-408 (1964)). Bailey et al. stated that the "mean antibody titer measured by an interfacial precipitation technique was 1:7000", but there was no attempt to produce or to measure antibodies that had specificity against cholesterol. The assay antigen consisted of the original conjugate, not cholesterol either alone or as part of another conjugate. Nowhere did Bailey et al. teach that they had induced antibodies to cholesterol, and they did not teach that antibodies to cholesterol could have been produced or that such antibodies might have played a role in the lowering of serum cholesterol levels or amelioration of atherosclerosis.
Bailey et al. observed a reduced hypercholesterolemia and less aortic plaque formation in the immunized animals that were fed a cholesterol-rich diet. However, in the absence of further information the antibody titer could have been entirely directed against the bovine serum albumin component and the cholesterol-albumin conjugate might simply have lowered cholesterol through nonspecific mechanisms, such as by nonspecific adsorption of serum cholesterol by the albumin. This latter explanation could be supported by the fact that albumin has a considerable degree of hydrophobicity and can be used as a reagent to promote solubility of cholesterol in an aqueous medium such as serum. The disclosure by Bailey et al. is too insufficient to draw any immunological conclusion regarding the role, if any, that antibodies to cholesterol may have played in the experimental results. It is probably because of this that Bailey et al. did not teach any such role.
Yet another embodiment of the invention relates to prevention and treatment of fungal infections in humans and animals. Among individuals who have reduced immunological function, for example, in those who have AIDS, cancer, trauma due to accidents or surgery, debilitative metabolic illnesses such as diabetes mellitus, persons whose blood is exposed to environmental microbes such as individuals having indwelling intravenous tubes, and even in some elderly individuals, fungal infections of blood and tissues can result in serious, even life-threatening, situations. Mortality rates in cancer patients who develop systemic fungal infections is very high. In other cases, fungal or fungus-like infections, usually introduced into the lungs through the air, are commonplace among large numbers of persons due to environmental exposures. Examples of the latter types of infections include: coccidioidomycosis which is indigenous to the San Joaquin Valley in California, and areas around Flagstaff, Ariz.; histoplasmosis, which is commonplace in the Midwest. Other common types of fungus, or fungus-like infections that can cause severe disseminated disease in immunocompromised patients include blastomycosis, crytococcosis, candidiasis, and mycobacterial infections such as tuberculosis.
It has been observed that fungi are the most common cause of nonbacterial infection in patients with leukemia and lymphoma, with Candida species and Aspergillus being the most common fungal species in cancer patients. These two infections are estimated to have a combined mortality of 20% (Lopez-Berestein, G., Mehta, R., Hopfer, R., Mehta, K., Hersh, E. M., and Juliano, R., Cancer Drug Delivery, 1:37-42, 1983). Certain other organisms that have parasitic properties, such as leishmaniasis, can mimic many of the disease-causing properties, behaviors, and pathologies of fungal infections.
A characteristic commonly shared by organisms that cause all of the above diseases is the presence of ergosterol as the predominant or sole sterol in place of cholesterol. Cholesterol is the major sterol that is found in mammalian cells and tissues. Ergosterol serves many of the physiological membrane-associated functions in these organisms that are served by cholesterol in mammals. Alteration of concentrations of cholesterol and ergosterol in lipid bilayer domains of plasma membranes has enormous effects on fluidity and permeability of the membranes, and the presence of ergosterol is essential for viability of certain microorganisms just as the presence of cholesterol is vital for viability of mammalian cells. The enormous importance of ergosterol is illustrated by the fact that ergosterol rather than cholesterol is the predominant sterol compound found in most plants. Cholesterol is rarely found in any membranes other than those of mammals, and cholesterol and ergosterol are rarely found in any species of bacteria.
Further, it is known that mammals concentrate antibodies in milk, including colostrum (the first post-partem milk produced) as well as subsequently produced milk. During cheese manufacturing, the antibodies may be concentrated in the whey. It has been previously demonstrated that immunization of dairy cows with antigens such as enterotoxic Gram negative E. coli or their CFA-1, CFA-2 pili results in the production of high concentrations of antibodies against the intact organisms and/or their infectious pili. The oral ingestion of milk products obtained from inoculated dairy animals, including whey, whey, concentrates, and other dairy products has been shown to result in the passive immunization of the recipient animal. The antibodies successfully survive and transit the stomach acidity and act in the gastrointestinal system to opsonize the ingested antigen, resulting in an antibody-organism complex that is harmlessly excreted.
What is needed are methods and compositions which can be used to vaccinate a human or an animal against sterols such as cholesterol or ergosterol. By vaccinating a human or animal against cholesterol, blood concentrations of cholesterol can be safely and inexpensively reduced. By vaccinating a human or animal against ergosterol, the human or animal can better resist infection by fungi. Further, by vaccinating a dairy animal against ergosterol the milk produced by the dairy animal will contain a high concentration of anti-ergosterol antibodies. Consequently, the milk and other dairy products derived therefrom will be resistant to fungus and may be used to passively immunize humans or animals.